1)Field of the Invention
The present invention relates to sensitive methods for detecting analytes such as explosives and drugs, which may be present in a sample at extremely low levels. The methods may be generally carried out in situ employing novel chemistry which is compatible with flow-cell technology and with time-scales and concentrations required for rapid and informative screening of large numbers of samples. The present invention also relates to novel compounds and apparatus for use with the methods disclosed.
The detection of plastic explosives, of drugs of abuse, of therapeutic agents, and of environmental pollutants is a field of growing importance in which there is a need for fast reliable, robust and simple detection methods. A major problem in this field is that the required analyte is often present in very low concentrations in a matrix such as a vapour or a body fluid. Thus, detection methods that are sensitive and rapid and which identify the analyte from other species are required.
2) Description of Related Art
One method of detection employs the detection of Raman scattering. Briefly, Raman scattering occurs when a light source irradiates a sample and scattered light is given off. Most of the light is scattered with the same frequency as that of the incident light but a weak component is scattered one vibrational unit different. The weak component is Raman scattering. By subtracting the frequency of the Raman scattered light from the frequency of the incident light, a vibrational spectrum characteristic of the molecule can be obtained. The light can then be detected in a suitable spectrometer, many of which are commercially available. The detection of Raman scattering is attractive since it uses visible or near infrared radiation to provide the exitation. Moreover, flexible and effective optics can be designed and water gives a weak signal so that detection in aqueous solution is possible. Further, the set of signals obtained gives a unique pattern from which a particular analyte can be identified. However, the main disadvantage of Raman scattering is that it is not sufficiently sensitive and is not therefore generally suitable for detecting analytes at extremely low concentrations, and fluorescence can interfere with detection.
The sensitivity of Raman scattering may however be improved. Firstly, if the analyte is adsorbed onto a suitably roughened metal surface of which silver and gold are the most widely used, then there is an interaction between the analyte and the surface electron waves on the metal (plasmons) which provide an enhancement in the intensity of the Raman scattering by a factor claimed to be 106. This technique is known as surface enhanced Raman scattering (SERS).
Another method of enhancing sensitivity is to use a dye with an absorption maximum at or close to the frequency of the exciting radiation. This enhanced scattering, called resonance Raman scattering, provides an increase in sensitivity of a few orders of magnitude in ideal cases. However, it is possible that fluorescence will interfere with this process.
Combining SERS and resonance Raman scattering: to give surface enhanced resonance Raman scattering (SERRS), provides more sensitivity and the conditions under which single molecule detection has been claimed. SERRS has been previously described, see for example Rodger, C. H., Smith, W. E., Dieht, G., Edmonson, M., J. Chem. Soc. Dalton Trans., 1996, 5, 791 and references therein to which the reader is directed for background information. Surprisingly, there is a widespread fluorescence quenching mechanism on the silver surface. This means that almost all dyes give SERRS rather than fluorescence on the surface enabling the use of more extensive derivatisation chemistry than is possible by fluorescence. Further, the SERRS active material scatters so strongly that the signal can be picked out from the background without the need for the removal of the matrix in which the sample is present so that separation procedures either before or after analysis are often not required.
However, the main disadvantage of SERRS is that it requires a specially labelled dye. To obtain reproducible results, this dye must also adhere strongly to a metal surface. Thus, although it is relatively easy to obtain very low atomolar detection limits, very variable results are often obtained and many molecules for which sensitive analysis would be of value are precluded from the method since they are not coloured and do not stick to the surface.
Recent patents describe the use of SERRS in DNA detection and in antibody detection (WO97/05280 & PCT/GB99/00588 respectively). In both these cases, a pre-formed molecule is used as the actual analyte. In DNA chemistry this is a label and in antibody chemistry it is a labelled antigen or ligand which is displaced from the antibody.
However, it will be appreciated that detection of analytes, such as explosives or drugs is not generally possible because of the lack of a suitable chromophore and/or the ability of the analyte to adhere to a metal surface. Moreover, it is desirable that detection of explosives or drugs can be effected quickly, for practical reasons, which is not generally possible using existing techniques.
It is an object of the present invention to obviate and/or mitigate at least one of the aforementioned disadvantages.